For over two decades, it has been generally assumed by clinical chemists that conjugated bilirubin (B.sub.c) in humans is chiefly a diglucuronide and that B.sub.c exists in the same molecular form in a variety of different body fluids which contain this metabolic product, for example, serum, bile, and the like. Owing to the unstable nature of B.sub.c, however, isolation of B.sub.c and specific molecular characterization of B.sub.c have been extremely difficult.
Definitive molecular characterization of B.sub.c has, to the knowledge of this inventor, been carried out for the first time by this inventor. See paper entitled "Human Conjugated Bilirubin--Isolation, Biosynthesis, and Molecular Characterization by Direct Spectroscopic Analyses" presented by T. W. Wu et al at the American Association for Clinical Chemistry 31th Annual Meeting in New Orleans, Louisiana, July 15-20, 1979. An abstract of this paper appears in Clinical Chemistry, Vol. 25, No. 6, p. 1137 (June, 1979). To determine the molecular structure of B.sub.c, B.sub.c was isolated by the bile extraction and isolation procedure reported by Lucassen, J., doctoral thesis, University of Utrecht, Netherlands (1961). Bile was chosen for the separation and isolation of B.sub.c because of the high concentration of B.sub.c which exists in bile. The principal molecular species of B.sub.c isolated from bile by the Lucassen procedure has now been found not to be a diglucuronide as many have previously speculated, but rather a diester having a molecular weight of 918.2 and containing one molecule of glucuronic acid and one of glucuronolactone as shown in Formula I: ##STR1## where R.sub.1 =glucuronic acid and R.sub.2 =glucuronolactone or
R.sub.1 =glucuronolactone and R.sub.2 =glucuronic acid PA0 Q represents a chemical bond(s) or a chemical group linking M.sup..sym. to A; PA0 M.sup..sym. represents a hydrophobic organic moiety containing a cation, preferably a quaternary ammonium or phosphonium group; and PA0 X.sup..crclbar. represents an acid anion such as a halide ion, for example, chloride or bromide; nitrate; methosulfate; p-toluenesulfonate; or an equivalent anion.
Recent work in the medical diagnostic field has indicated that the presence and concentration of B.sub.c in body fluids is of diagnostic significance, particularly in dealing with the treatment of certain jaundice conditions, e.g., in obstructive jaundice the small amount of B.sub.c normally present in adult human serum becomes elevated to form a larger proportion of the total bilirubin content thereof. The total bilirubin content consists of the sum of B.sub.c and the more familiar, predominant form of bilirubin referred to as unconjugated bilirubin (B.sub.u).
To develop assay methods for bilirubin which selectively detect both B.sub.c and B.sub.u, it became apparent that improved methods for separating and isolating B.sub.c and/or B.sub.u from aqueous liquids, particularly those containing mixtures of both B.sub.c and B.sub.u, are necessary. This is because of the need to have B.sub.c and B.sub.u standards effective to calibrate assays for B.sub.c and B.sub.u. At the present time, B.sub.u standards that can be purchased typically come from nonhuman bile sources, e.g., cow or ox, while B.sub.c standards are not available from either human or nonhuman animal sources. Various artificial standards have been used for bilirubin such as ferric thiocyanate, cobalt sulfate, potassium permanganate and the like. However, as reported by Winkelman, J. et al, Clinical Chemistry--Principles and Technics, R. J. Henry, D. C. Cannon, and J. W. Winkelman, Eds., Harper & Row Publishers, New York, 2nd Ed., 1974, pp 1038-1070, these artificial standards do not work well in spectrophotometric assays, because their absorption curves are not the same as that of bilirubin.
Although one could attempt to obtain a B.sub.c standard by use of the above-referenced Lucassen procedure, this procedure is difficult to perform. In addition, the Lucassen procedure provides little or no stabilization of B.sub.c during the isolation and extraction process, and therefore the B.sub.c has a pronounced tendency to degrade during the process. Moreover, the Lucassen procedure, although useful on bile which contains relatively high concentrations of B.sub.c and low concentrations of protein to which bilirubin readily binds, has never to the knowledge of the inventor been successfully applied to other aqueous biological liquids. For example, the Lucassen procedure has been found unsuccessful when applied to serum which contains relatively low amounts of B.sub.c and high concentrations of protein. Accordingly, a new method for separating and isolating B.sub.c and/or B.sub.u from bile as well as other aqueous liquids such as serum would be highly desirable.
Once separated and isolated, B.sub.c and/or B.sub.u can be employed for numerous clinical purposes such as in the manufacture and preparation of various standards, i.e., reference compositions, such as calibrators and controls, used in the assay of bilirubin contained in biological fluids such as serum, urine, cerebrospinal fluid, bile and the like.